FR2466252A2 - Cytotoxic prods. - in which an antibody is linked to a chain of ricin purified by chromatography - Google Patents

Abstract

Cytotoxic prod. is prepd. by forming a covalent disulphide bridge between a cytotoxic cpd., the A chain of ricin, and antibody, and immunoglobulin or a specific immunoglobulin fraction of a given antigen, according to the parent patent. The A chain of ricin is purified by chromatography to give a crystalline prod. Cytotoxic prods. as described in the parent patent but in which the antibody is either fragment F (ab)'2 of anti-DNP antibody, or it is IgM anti-Thy 1.2 are also new. This is an addn. to FR 7827838. The purified chain A is free from non-specific toxicity to the cell system or animal organism and so the specificity of the prod. is solely that of the antibody used.

Description

 The present invention relates to novel products having in particular anticancer properties.

 In the patent application filed September 28, 1978 under the number 78 27838, the Applicant has described anti-cancer products consisting of coupling a cytotoxic agent channe A of Ricin with an immunoglobulin specific for antigens carried by cancer cells.

These products, referred to as "conjugates", were prepared by forming a disulfide bridge between the free thiol group optionally activated as a disulfide form of the A ring of the Ricin and an immunoglobulin into which one or more thiol groups or one or more activated disulfide groups. The subject of this addition is various improvements to the method described in the main application as well as the preparation of new conjugates according to this method.
The first improvement concerns the preparation of the channe A de la Ricine. The method indicated in the main application led to a diluted solution of Ricine channe A. It has been found that by subjecting this solution to a concentration stage on a CMSepharose @ combining the effects of ion exchange and affinity, a solution having both a high concentration and a very high purity can be obtained. In addition, such a solution leaves deposit by cooling the channe A in crystallized form.

 The A chain thus prepared is free of nonspecific toxicity towards cellular systems or animal organisms and can therefore be used for the preparation of conjugates whose specificity is strictly provided by the antibody.

In addition, the present application describes the preparation of new conjugates. These new conjugates are of different types
conjugated between the channel A of Ricin and the anti-DNP antibody obtained by binding to the antibody of a reagent of the disulfide type different from that used in the main application. In this, the reagent used was 2- (2-pyridyl disulfanyl) propionic acid, whereas in the present application it is ((2-pyridyl-disulfanyl) -propionylamino] -6-hexanoic acid;
conjugated between the Ricine A chain and an antibody F (ab) '2 fragment. These F (ab)' 2 antibody fragments containing the recognition sites are sufficient to ensure the specificity of the conjugates.
conjugated between the channe A of Ricine and an immunoglobulin of the M type (IgM) whose antibody specificity is the recognition of the allelic form 1,2 of the alloantigen Thy carried especially by the cells of certain lymphotogenic cancerous Lture, 209, 5022, 521 (1966)].

 The following examples illustrate the invention.

EXAMPLE 1
Concentration of the channe A of Ricine.

 The chain A obtained in dilute solution according to Example 2 of the main patent is deposited on a chromatography column of 16 mm internal diameter containing 10 ml of Carboxymethyl Sepharose # equilibrated with the same buffer. Under these conditions, the channe A fixes itself; it is then eluted with the TPE buffer.

Recovery is quantitative.

 A d.Sptt of 150 mg of channe A elutes fractions at the average concentration of 10 mg / ml channe A, representing a total of 90% of the amount deposited on the column.

 The concentrated solution of -chaine A thus obtained has a higher purity than that resulting from the application of the protocol described in the main application, as will be demonstrated in the chapter "Results".

Obtaining cat-crystals A.

 A chain A solution of concentration equal to 10 mg / ml is abandoned at 40C. After a few days crystals appear whose analysis, after redissolution, reveals that they possess the same physicochemical properties (molecular weight, isoelectric point) and biological properties (inhibition of proteosynthesis) as the A chain in solution from which they arise.

EXAMPLE 2
Preparation of the conjugate obtained from anti-DNP antibodies substituted with an activated disulfide group and a Ricine A chain.

a) [(2-pyridyl-disulfanyl) -3-propionyl amino] -6 hexanoyl.

This acid is obtained in 2 steps from (2-pyridyl-3-disulfanyl) propionic acid without purification of the intermediate product.

 0.430 g of the (2-pyridyl disulfanyl) propionic acid obtained as described in Example 6 a) of the main application is dissolved in 3 ml of pyridine. The solution is cooled in an ice bath. 0.230 g of powdered N-hydroxysuccinimide and 0.453 g of dicyclohexyl carbodiimide are also added to this solution. The mixture is stirred for 20 h at 40C. The reaction medium is filtered to remove the precipitate of dicyclohexylurea formed, the precipitate is washed with pyridine. The wash solution is added to the filtrate and then the pyridine is evaporated to dryness under high vacuum. The residue obtained is taken up in ethyl acetate and a new precipitate of dicyclohexylurea is formed; it is removed by filtration. The ethyl acetate is evaporated to dryness under high vacuum and the washing operation with ethyl acetate is repeated twice. 0.610 g of the expected ester are thus obtained.

 The product obtained is dissolved completely in 5 ml of tetrahydrofuran. To this solution are added 0.288 g of 6-amino-caprotic acid in solution in 2.5 ml of water. Triethylamine is added in stoichiometric amount relative to 6-amino-caproic acid (0.222 g) to maintain the pE at 7.0. the reaction is continued for 20 h at 40 ° C. with stirring. The reaction medium is evaporated to dryness under high vacuum. The residue is taken up in 10 ml of water.

The medium is acidified to pH 4.5 by addition of acetic acid.

A precipitate forms which is recovered by decantation and then solubilized in ethyl acetate. The solution is dried with magnesium sulfate and filtered and evaporated to dryness under high vacuum.

 0.600 g of the product to be purified is obtained. The purification is carried out by partition chromatography on a silica column, under conditions defined by analysis of the product by thin layer chromatography in acetone-chloroform-acetic acid (15-80-5).

 0.410 g of the product obtained are deposited on a column of internal diameter 2 cm containing 160 ml of silica gel (nO 60, 60-230 mesh MERCK) equilibrated with chloroform. The elution is carried out in a discontinuous gradient of methanol in chloroform. The pure product is eluted with the mixture at 2% of methanol. The solvents are evaporated to dryness under high vacuum.

 The product obtained, pale yellow and of a ponderous consistency, is characterized by its nuclear magnetic resonance spectrum.

b) Preparation of activated anti-DNP antibodies.

 To 9 ml of the anti-DNP antibody solution at a concentration of 11.4 mg / ml in the TPE buffer is added 9 ml of a 2: 1 (v / v) water / t.butanol mixture containing 3 mg of 1-ethyl-3-dimethylpropyl-3-carbodiimide and 13.7 mg of (2-pyridyl-disulfanyl) -3-propionylamino] -6-hexanoic acid are then carried out as described in US Pat. example 6 of the main application.

 The assay of the activated antibodies indicates an average substitution rate of 0.6 activating group per mole of antibody.

 The antibody solution thus obtained is concentrated by ultrafiltration to 11.1 mg / ml.

c) Conjugate
48.8% of the modified antibodies are reacted with 35.6 mg of the Ricin A chain at 7> 9 mgXml in the TPE buffer.

the preparation is carried out under the conditions already written in example 6 of the main application. The average coupling ratio obtained is 0.5 A chain per mole of antibody. Purification of the conjugate by filtration is carried out as described in Example 6 of the main patent.

EXAMPLE 3
Preparation of the conjugate obtained from the F (ab) '2 fragment of the anti-DNP antibody and the Ricin A chain.

 a) Preparation of the F (ab) fragment of the anti-DNP antibody.

2
The antibody solution (Example 4 of the main application) is dialyzed against the 120 mM acetate buffer pH 4.7. To 45 ml of this solution containing 500 mg of antibody, 3 ml of a solution of pepsin at a concentration of 10 mg / ml in the same buffer (Pepsin EC 3.4.4.1 pork, twice crystallized, with 3200 u / mg SIGMA). Incubation is maintained for 20 h at 370C; the progress of the hydrolysis is verified by electrophoresis in the presence of sodium dodecyl sulphate [J. Biol. Chem. 244, 4406-4412, (1969)] by disappearance of the 150000 dalton band corresponding to the unhydrolyzed antibody.

 The hydrolysis is stopped by adjusting the pH of the reaction medium to 7.0 with a 1N sodium hydroxide solution, then the solution is centrifuged and the insoluble material is removed. The centrifugation supernatant is deposited on a column of 50 mm internal diameter containing 1800 ml of Sephadex G, equilibrated with 100 mM phosphate buffer pH 7.0.

The elution is carried out with the same buffer: the first peak obtained is collected in two fractions; the first (200 ml) contains 70 mg of F (ab) 'fragment of the antibody, the second (100 ml)
2 tins, contains 70 mg of a mixture of F (ab) '2 fragment and a contaminating fragment of molecular weight 50000 dalton. The second peak contains protein fragments of average molecular weight 10000 Dalton it is discarded.

 The F (ab) '2 fragment obtained pure in the first fraction of the first peak is concentrated by ultrafiltration (protocol described in Example 1 of the main application) to the concentration of 16 mg / ml and stored at -200C.

The F (ab) '2 fragment thus obtained has the following characteristics. Molecular weight: 95000 dalton (determined after calibration of
R
Sephadex G 150 gel).

. Isodlectric weight: between pH 7.7 and 8.8 (obtained by
isoelectric focusing on LKB plate).

 b) Preparation (ab) 'of the activated F (ab)' fragment.

2
15.3 ml of a water / tert-butanol 2/1 (v / v) solution containing 17.3 mg are added to 15 ml of the F (ab) '2 solution at a concentration of 15.6 mg / ml. of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50.4 mg of 2-pyridyl-disulfanyl-3-propionic acid. The mixture is incubated for 20 h at 300C. The solution is then dialyzed continuously against the TPE buffer at 40 ° C. for 23 hours at a flow rate of 500 ml / h.

The spectrophotometric determination at 343 nm of the pyridine thione-2 released by exchange with reduced glutathione shows that the activation of the F (ab) '2 fragment led to the average substitution rate of 1.5 activating groups per mole of F ( ab) '2.

 The activated F (ab) '2 solution thus obtained is concentrated by ultrafiltration to 10 mg / ml.

 (c) Conjugate.

To 13.8 ml of the 10 mg / ml solution in F (ab) 'activated
2 obtained as described above are added 13.8 ml of 8 mg / ml Ricine A chain solution in TPE buffer.

 The mixture is incubated at room temperature for 24 hours under a nitrogen atmosphere and protected from light. the solution is then centrifuged at 27,000 x g at 40C for 10 min and the supernatant recovered. The determination of the pyridine thione-2 released during the reaction indicates that the average coupling ratio is 1.2 equivalent of A chain per mole of F (ab) '2.

 The solution is deposited on a column of 50 mm inside diameter containing 1730 ml of Sephadex G 200. The elution is carried out with the TPE buffer. The fractions are collected in a volume of 8.4 ml and the analysis of the fractions is carried out as described in Examples 5 and 6 of the main application. The conjugate proceeds in a peak.

The fractions composing the peak of the conjugate are grouped into three solutions which are respectively
- Solution f: front of the peak
Solution g: central portion of the peak;
- Solution h: tail of the peak.

The analysis of the solution g by the determination of its inhibitory activity of the proteosynthesis (test 1) and the determination of the proteins gives the following results:

<tb><SEP> / ug <SEP> of <SEP> string <SEP> A <SEP> Equivalent <SEP> of
<tb><SEP> paired <SEP> to <SEP> F (ab) '2 <SEP> string <SEP> A <SEP> by
<tb><SEP> by <SEP> ml <SEP> of <SEP> solution <SEP> mole <SEP> of <SEP> F (ab) '2
<tb> Solution <SEP> g <SEP> 148 <SEP> 1,4
<tb> d) Conjugate concentration.

 In a first step, the concentration is carried out by ultrafiltration: thus, 255 ml of the conjugate solution (1481 μg / ml in A chain) are concentrated to 381 μg / ml. The solution is then diluted 10 times in distilled water to lower the ionic strength of the medium.

 The solution obtained (400 ml at 38 μg / ml A chain) is deposited on a column of internal diameter 9 mm containing 5.7 ml of CM Sepharose (equilibrium with 10 mM phosphate buffer pH 6.5 mixed with disodium salt). 1N ethylenediaminetetraacetic acid.

 The deposit is made at a rate of 80 ml / h. Under these conditions, all the conjugate is fixed on the column. At the end of the deposition, 4.5 ml of the same buffer is allowed to pass and then the conjugate is eluted with the TPE buffer at the same flow rate.

 you conjugate comes out in a single peak. The tail of the spike, containing little protein, is discarded; the remaining fractions are pooled and the A chain concentration is determined.

The assay indicates that the concentration operation on
CM Sepharose (B) made it possible to increase the concentration of the A chain of the conjugate to 3.6 mg / ml. The operation is quantitative and the fractions making up the 3.6 mg / ml solution represent a total of 76% of the amount of conjugate deposited on the column.

EXAMPLE 4
Preparation of the conjugate obtained from the antibody
IgM anti-Thy 1.2 and Ricine A chain.

a) Preparation of Anti-Thy 1.2 IgM Antibody
Pure IgM is prepared from the ascites fluid obtained from OLAC LTD (Thy-l.2 F7 D5 monoclonal IgM cytotoxic antibody).

 To 50 ml of ascites fluid containing the antibody are added 15 g of ammonium sulfate powder. After dissolution, the mixture is incubated for 1 h at 40C then the whole is centrifuged for 20 min at 17000 x g.

The centrifugation supernatant is discarded. The pellet is dissolved in 20 ml of 100-mM phosphate buffer pH 7.0 and the solution is dialyzed continuously against 10 1 of the same buffer. The solution obtained is deposited on a column of 50 mm internal diameter containing 1800 ml of Sepharose 6B # equilibrated with the 100 mM phosphate buffer pH 7.0. The solution is carried out with the same buffer: the first peak obtained corresponds to aggregates of IgM. The second peak obtained is collected in two fractions: the first fraction (from the front to the top of the peak) is composed of pure IgM; the second fraction contains a mixture of IgM and a contaminating protein of molecular weight evaluated at about 660000 daltons. This second fraction is treated again with ammonium sulphate and the precipitate obtained is centrifuged and then redissolved in the 100 mM phosphate buffer pH 7.0. The solution obtained is deposited on the column of
Sepharose 6B (X) suitably washed after the first operation.

 Elution with the 1130 mM phosphate buffer pH 7.0 gives rise, as in the first operation, to the appearance of two peaks. The second peak obtained contains the pure I M. Each of the two Sepharose 6B R filtration operations thus made it possible to obtain a fraction containing the pure IgM. These two fractions are combined and then the whole is treated with ammonium sulphate at the final concentration of 300 mg / ml, at 40C; the preparation is centrifuged for 20 min at 17000 x g and then the pellet is taken up in 100 mM phosphate buffer pH 7.4 supplemented with 400 mM sodium chloride.

 The set of operations makes it possible to prepare 8 ml of a solution containing 6.4 mg / ml of pure IgM. Electrophoresis analysis in the presence of sodium dodecyl sulfate was used to characterize the fractions; the pure IgM thus obtained has two very close bands of equal intensities, corresponding to very close molecular weights of 900,000 daltons.

b) Preparation of the activated anti-Thy 1.2 antibody.

 To 7.6 ml of the IgM solution at the concentration of 7.0 mg / ml is added 0.46 ml of a 1/5 (v / v) water / tert.butanol mixture containing 2.9 mg of 1-ethyl-3- (3-dimethyl-3-propyl) carbodiimide and 7.7 mg of (2-pyridyl-disulfanyl) -3-propionic acid. The mixture is incubated for 20 h at 300C. The solution is then dialyzed continuously against the 100 mM phosphate buffer pH 7.4 supplemented with 400 mM sodium chloride and the disodium salt of 1 mM ethylenediaminotetraacetic acid. Dialysis is continued for 23 h at 4 ° C. at a flow rate of 500 ml / h. The solution is then centrifuged at 27000 × g at 40 ° C. for 10 min and the supernatant recovered. The spectrophotometric measurement at 343 nm of the pyridine thione-2 released by exchange with the reduced glutathione shows that the activation of the antibody has led to average substitution rate of 13 activating groups per mole of IgM.

(c) Conjugate.

 The A chain solution obtained as described in Example 1 is continuously dialyzed against 100 mM phosphate buffer pH 7.4, supplemented with 400 mM sodium chloride and disodium salt of 1 mM ethylenediaminotetraacetic acid; dialysis is conducted at a flow rate of 500 ml / h for 20 h at 40C.

 6.8 ml of the dialyzed A-chain at a concentration of 7.4 mg / ml are added to 6.8 ml of the activated IgM solution obtained as described above. The mixture is incubated for 24 hours at room temperature under a nitrogen atmosphere and protected from light. assay for pyridine thione-2 released during the reaction indicates that the average coupling rate is 9.7 equivalents of A-chain per mole of IgM.

 The solution is deposited on a column of inner diameter 50 mm containing 1727 ml of Sepharose 6B (E) equilibrated with the conjugation buffer. Elution is conducted with the same buffer.

The fractions are collected in a volume of 3.7 ml and the analysis of the fractions is carried out as described in Examples 5 and 6 of the main application. The conjugate proceeds in two peaks; the first corresponds to the conjugate of average molecular weight greater than that of IgM; the second peak corresponds to the conjugate of average molecular weight approximately equivalent to that of the IgM. The central part of this peak (solution i) is retained.

The conjugate composition of the solution i corresponds to the following characteristics

<SEP> / ug <SEP> of <SEP> string <SEP> A <SEP> Equivalents <SEP> of
<tb><SEP> paired <SEP> a <SEP>1'IgM<SEP> string <SEP> A
<tb><SEP> with <SEP> ml <SEP> of <SEP> solution <SEP> with <SEP> mole <SEP> of IgM
<tb> Solution <SEP> i
<Tb>
The conjugates according to the invention as well as the compounds used in the preparation of said conjugates have been studied with regard to their biological properties and particularly their anticancer action.

 The different tests used are indicated below.

TESTS IN VITRE 1 - Inhibition of proteosynthesis in acellular model;
Test described in the main application.

2 - Inhibition of proteosynthesis in cell model
In the case of anti-DNP specific conjugate, this test was described in the main application.

 For the anti-Thy 1.2 specific conjugate, the cells used are: WEHI-7 line (Balb / C mouse lymphoma) obtained from SALK INSTITUTE-SAN DIEGO (California). These cells do not undergo any labeling step because they naturally carry Thy 1.2 antigen. on their surface. The incubation with the conjugate and the measurement of the proteosynthesis rate proceed as described above.

RESULTS
Chain A: The concentrated solution of chain A in the TPE buffer, prepared as described in Example 1, is subjected to the assay of its cell-free proteosynthesis inhibition activity (test 1) and on a cellular system (test 2 ).

The activity of the A chain in the test 1 is identical to that measured on the A chain preparation described in the main application, which indicates that the intrinsic biological property of the A chain is not changed by the step of focus on
CM Sepharose
The results of the assays according to test 2 are presented in the table below

<tb><SEP> Expression <SEP> Report <SEP> of acti
<tb><SEP> of <SEP> Ricin <SEP> String <SEP> A <SEP> vited <SEP> Ricin /
<tb><SEP> the activity <SEP> string <SEP> A
<tb> String <SEP> A <SEP> prepared
<tb> as <SEP> describes <SEP> in <SEP> the <SEP> 8 <SEP>
<tb> request <SEP> main <SEP> 5.10 <SEP> 1250
<tb> (reminder) <SEP> IC50 <SEP> in <SEP>4.10'11<SEP>
<tb><SEP> mole / liter
<tb> String <SEP> A <SEP> having <SEP> sustained <SEP> -8
<tb> the <SEP> concentration <SEP> on <SEP> 68.6.10 <SEP><SEP> 17150
<tb> C. <SEP> M. <SEP> Sepharose
<Tb>
These results indicate that the concentration step on
CM Sepharose makes it possible to further reduce the non-specific toxicity of the A chain by significantly increasing the purity of the preparation. The purissime chain thus produced is devoid of non-pacific toxicity towards a cellular system; this is clearly shown by the ratio of activity between the Ricine and the A chain in this test, which is greater than 10,000.

Comprehensive A-chain uptake assays were performed using successively excess Hela cells and red blood cells contacted with the A-chain and measuring cell A cell toxicity in the supernatant-absorption ( test 2)
the cellular toxicity of the A-chain solution prepared, as described in the main application, is lowered by absorption and thus reduced to the level of the toxicity of the C14-concentrated chain A solution. Sepharose and not absorbed;
on the other hand, the toxicity of the preparation concentrated on CM Sepharose is no longer diminished by the absorption tests, which confirms the total absence of affinity of the thus prepared A chain towards the plasma membranes of the cells and, by As a consequence, the extreme degree of purity obtained.

Conjugates:
The specific toxicity of the conjugates was assayed in Test 2 and the results shown in Figures 1 and 2.

These figures respectively represent
for FIG. 1, the inhibition of the incorporation of
C-leucine in Hela and Hela-TNP cells; in
14
ordered the incorporation of C-leucine into
percent and on the abscissa the concentrations (nM) of
chain A; the various curves represent the re
results obtained for
11
. Ricin: IC 50 (in A chain) 5.5 M. (1)
. the chain A: IC50 (in A chain) 9.10 7 M. (2)
. conjugate (solution g)
on Hela: IC50 (in A chain) not determinable (3)
(greater than 10-5 M) on Hela-TNP: IC50 (in A chain) 4.5.10 -9 M. (4)
for FIG. 2, the inhibition of the incorporation of
14C-leucine in wEHI-7 cells; on the ordinate
14
carried the incorporation of C-leucine in percent and
on the abscissa the concentrations (nM) of chain A; the
various curves represent the results obtained for -11
. Ricin: IC 50 (chain A) 2,4.10 M. (1)
the chain A: IC 50 (in chain A) 8.1 × 10 -7 M. (2)
. Pure IgM (IgM concentrations are reported
at the same scale as the chain concentrations A)
not determinable. (3) Conjugate -11
(solution i): IC 50 (as A chain) 5.2 × M.

 The conjugate formed from the Ricin A chain and the anti-DNP IgG F (ab) '2 fragment exhibits high toxicity to IL-TNP cells (Figure 1).

 This toxicity is associated with the specificity of action since the ratio of the activities towards Hela cells bearing and not carrying the TNP is greater than 2000. The use of the antibody fragments carrying the antigen binding sites thus makes it possible to ensure the effectiveness and specificity of the conjugates that come from it.

 The conjugate formed to suffer from Ricin A-chain and anti-DNP IgG, using 2- (2-pyridyl-disulfanyl) propionylamino] -6-hexanoic acid as the activator of IgG, has a toxicity and specificity identical to those of the conjugate prepared in Example 6 of the main application.

 The establishment of the disulfide bridge between IgG and A chain via this type of activator molecule thus results in efficient and specific conjugates.

 The conjugate formed from the A chain and IgM srfti-Thy 1.2 has a very high toxicity towards the cancer cells carrying this antigen (FIG. 2). The specificity of action is very large (greater than 10,000 between the activity of the conjugate and that of the A chain).

 It has thus been demonstrated that the establishment of the disulphide bridge between the A chain and an antibody specific for an antigen naturally carried by cancerous cells makes it possible to destroy the cancer cells with a very high efficiency and a high specificity of action.

TEST IN VIVO
The conjugate prepared in Example 4 was tested for its anticancer activity in vivo.

The cancer cells used are of the line
WEHI 22.1 (obtained from SALK INSTITUTE-SAN DIEGO - California) from a Balb / C mouse lymphoma. They are injected into Balb / C mice obtained from B051OLTGAARD (Denmark) and maintained from birth and for the duration of the test in a zone free from specific pathogenic organisms (SPF).

In the test, the WEHI 22.1 cells maintained in continuous culture are collected on the third day after transplanting the culture, washed by centrifugation and resuspended in isotonic phosphate buffer (PBS) at a concentration of 12 × 10 6 cells / ml.
0.2 ml of the suspension is injected intraperitoneally to the mice previously randomly distributed in their cages and identified individually by tagging the legs.

 In mice not treated with the conjugate, the intraperitoneal injection of wEHI 22.1 cells is followed by the development of these cells into tumors dispersed in the peritoneum with formation of ascites. The development of cells is accompanied by an increase in body weight. Ascites is detected by observing the increase in abdominal volume.

 One hour after injection of the cells, the animals treated with the conjugate intraperitoneally receive 1 ml of solution of the conjugate (solution i, 23.7 μg / ml in A chain) previously extensively dialyzed against the PBS buffer and then sterilized by filtration.

The batches of control animals receive 1 ml of PBS buffer containing no conjugate.

 On the seventh day after inoculation of the cells, the treated animals receive a second injection of conjugate under the same conditions and the control animals a second injection of PBS buffer.

 The test results appear in Figures 3 and 4.

Figure 3 depicts the chronology of the occurrence of ascites in treated and untreated animals; the percentage of animals with ascites was plotted on the y-axis and the number of days after inoculation of WEHI cells 22.1. The solid curve represents the results obtained on treated animals and the curve in broken lines the results obtained on untreated animals.

 Figure 4 shows the average increase in body weight (in g) (the baseline corresponds to the weight of the animals before inoculation) as a function of the number of days after inoculation of WEHI cells 22.1. This change in weight is expressed in relation to the weight of these same animals 3 days before the inoculation of the cells. The solid curve corresponds to the results obtained on treated animals and the curve in broken line the results obtained with untreated animals.

The linear fit is done using the least squares method. The interpretation of the results is given in the following table

<tb><SEP> Coefficient <SEP> Slope <SEP> Variation <SEP> type
<tb><SEP>
<tb> Animals <SEP> of <SEP> controls <SEP> Slope <SEP> 0.54 <SEP><SEP> type <SEP>
<tb> Animals <SEP> treated <SEP> t <SEP>0> 89 <SEP> 0.64 <SEP>0> 04 <SEP>
<Tb>
Figure 3 shows an inhibition of ascites formation in animals treated with the conjugate; this inhibition results in a shift in the appearance of ascites.

 Figure 4 also shows an inhibition of body weight increase in the animals treated with the conjugate compared with control animals.

These observations are validated by the statistical analysis of the results
the inhibition by the conjugate of the formation of ascites is significant at the confidence level of 1% (test X2)
the inhibition by the conjugate of the weight increase is significant at the 5% confidence level (comparison test of the slopes of the linear adjustments),
The in vivo test makes it possible to demonstrate the anticancer activity of the conjugate formed from the Ricine A chain and the anti-Thy 1.2 IgM. This activity is revealed by the inhibition of the development of WERI 22.1 cancer cells injected into the Balb / C mouse.

Claims (2)

 1 - Process for the preparation of cytotoxic products by causing the formation of a covalent bond by means of a disulfide bridge between a cytotoxic compound, the A chain of the Ricin, and an antibody, immunoglobulin or immunoglobulin fraction specific for a given antigen, carried by the cells that are to be destroyed, characterized in that the A chain of the Ricine is purified by chromatography until, after cooling, crystallized A chain is obtained.  2 - Novel cytotoxic products consisting of conjugated compounds in which the A chain of the Ricine is coupled by a disulfide bridge to a suitable protein structure, especially an antibody, characterized in that said antibody is chosen from:  the F (ab) '2 fragment of the anti-DNP antibody;  IgM anti-Thy 1.2.